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- PDB-6lvd: Structure of Dimethylformamidase, tetramer, Y440A mutant -

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Basic information

Entry
Database: PDB / ID: 6lvd
TitleStructure of Dimethylformamidase, tetramer, Y440A mutant
Components
  • N,N-dimethylformamidase large subunit
  • N,N-dimethylformamidase small subunit
KeywordsHYDROLASE / ab polypeptide / mononuclear iron / amidohydrolase / tetramer
Function / homologyN,N-dimethylformamidase / N,N-dimethylformamidase activity / N,N-dimethylformamidase beta subunit-like, C-terminal / N,N-dimethylformamidase beta subunit-like, C-terminal / Concanavalin A-like lectin/glucanases superfamily / Concanavalin A-like lectin/glucanase domain superfamily / metal ion binding / N,N-dimethylformamidase large subunit / N,N-dimethylformamidase small subunit
Function and homology information
Biological speciesParacoccus sp. SSG05 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsArya, C.A. / Yadav, S. / Fine, J. / Casanal, A. / Chopra, G. / Ramanathan, G. / Subramanian, R. / Vinothkumar, K.R.
Funding support India, 3items
OrganizationGrant numberCountry
Department of Biotechnology (DBT, India)DBT/PR12422/MED/31/287/2014 India
Department of Biotechnology (DBT, India)BT/PR5081/INF/22/156/2012 India
Science and Engineering Research Board (SERB)SB/S2/RJN-094/2017 India
CitationJournal: Angew Chem Int Ed Engl / Year: 2020
Title: A 2-Tyr-1-carboxylate Mononuclear Iron Center Forms the Active Site of a Paracoccus Dimethylformamidase.
Authors: Chetan Kumar Arya / Swati Yadav / Jonathan Fine / Ana Casanal / Gaurav Chopra / Gurunath Ramanathan / Kutti R Vinothkumar / Ramaswamy Subramanian /
Abstract: N,N-dimethyl formamide (DMF) is an extensively used organic solvent but is also a potent pollutant. Certain bacterial species from genera such as Paracoccus, Pseudomonas, and Alcaligenes have evolved ...N,N-dimethyl formamide (DMF) is an extensively used organic solvent but is also a potent pollutant. Certain bacterial species from genera such as Paracoccus, Pseudomonas, and Alcaligenes have evolved to use DMF as a sole carbon and nitrogen source for growth via degradation by a dimethylformamidase (DMFase). We show that DMFase from Paracoccus sp. strain DMF is a halophilic and thermostable enzyme comprising a multimeric complex of the α β or (α β ) type. One of the three domains of the large subunit and the small subunit are hitherto undescribed protein folds of unknown evolutionary origin. The active site consists of a mononuclear iron coordinated by two Tyr side-chain phenolates and one carboxylate from Glu. The Fe ion in the active site catalyzes the hydrolytic cleavage of the amide bond in DMF. Kinetic characterization reveals that the enzyme shows cooperativity between subunits, and mutagenesis and structural data provide clues to the catalytic mechanism.
History
DepositionFeb 2, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 3, 2020Provider: repository / Type: Initial release
Revision 1.1Jul 15, 2020Group: Database references / Category: citation / Item: _citation.title
Revision 1.2Dec 16, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Mar 27, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: N,N-dimethylformamidase large subunit
B: N,N-dimethylformamidase small subunit
C: N,N-dimethylformamidase large subunit
D: N,N-dimethylformamidase small subunit
E: N,N-dimethylformamidase large subunit
F: N,N-dimethylformamidase small subunit
G: N,N-dimethylformamidase large subunit
H: N,N-dimethylformamidase small subunit


Theoretical massNumber of molelcules
Total (without water)409,3348
Polymers409,3348
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy, At low concentration, the molecules can disassemble to form dimer containing 2x alpha+beta
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area33400 Å2
ΔGint-170 kcal/mol
Surface area112790 Å2

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Components

#1: Protein
N,N-dimethylformamidase large subunit


Mass: 86249.664 Da / Num. of mol.: 4 / Mutation: Y440A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Paracoccus sp. SSG05 (bacteria) / Gene: dmfase2 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): star / References: UniProt: I6NT79, N,N-dimethylformamidase
#2: Protein
N,N-dimethylformamidase small subunit


Mass: 16083.823 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Paracoccus sp. SSG05 (bacteria) / Gene: dmfase1 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): star / References: UniProt: I6NWZ0, N,N-dimethylformamidase

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Dimethylformamidase / Type: COMPLEX / Details: Tetramer, 2x(a2b2) / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.4 MDa / Experimental value: NO
Source (natural)Organism: Paracoccus sp. SSG05 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21 DE3 (Star)
Buffer solutionpH: 7.2
Buffer component
IDConc.NameBuffer-ID
150 mMTris-Cl1
2200 mMNaCl1
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Sample at higher salt exists mostly as tetramer
Specimen supportDetails: Glow discharge was performed with Quorum glocube at 25mA, for 90 s.
Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R0.6/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 291.15 K
Details: Blot force was 0 and blotting was done for 3.5 seconds

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 75000 X / Calibrated magnification: 130841 X / Nominal defocus max: 4531.4 nm / Nominal defocus min: 1216.5 nm / Calibrated defocus min: 1216.5 nm / Calibrated defocus max: 4531.4 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 77.7 K / Temperature (min): 77.7 K
Image recordingAverage exposure time: 60 sec. / Electron dose: 27.5 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 443
Details: A total of 25 frames were saved from the 60 second exposure, resulting in ~1.1 electron/frame
Image scansSampling size: 14 µm / Width: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategoryDetails
2EPU1.9image acquisition
4Gctf1.06CTF correctionGctf was used to estimate CTF values
7Coot0.9-premodel fitting
9PHENIX1.13model refinement
10RELION3initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
CTF correctionDetails: CTF correction was done with Relion, during refinement and reconstruction
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 51556
Details: Two rounds of 2D classification was performed prior to angular assignment and reconstruction
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 18645 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 52.5 / Protocol: BACKBONE TRACE / Space: REAL

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